Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD

Hypoxia alters the biological functions of skeletal muscle cells to proliferate and differentiate into myotubes. However, the cellular responses of myoblasts to hypoxia differ according to the levels of oxygen and the types of cells studied. This study examined the effect of hypoxia (1% oxygen) on bovine satellite cells. Hypoxia significantly increased the proliferation of satellite cells cultured in a growth medium. In addition, the levels of PCNA, cyclin D1, cyclin-dependent kinase-1 (CDK1) and CDK2 expression were increased. Hypoxia facilitated the formation of myotubes as well as the stimulation of MyoD, myogenin, and myosin heavy chain (MHC) expression in differentiating medium (DM) cultures. In particular, satellite cells cultured under hypoxic/DM conditions showed increased p21 expression but not p27. The transfection of satellite cells with antisense MyoD oligonucleotides resulted in a decrease in the MHC, myogenin, MRF4 RNA and protein levels with the concomitant decrease in fused cells to levels similar to those observed under normoxia/DM conditions. This indicates that MyoD up-regulation is closely associated with hypoxia-stimulated myogenic differentiation. In conclusion, hypoxia stimulates the proliferation of satellite cells and promotes their myogenic differentiation with MyoD playing an important role.     

Myogenic gene expression at rest and after a bout of resistance exercise in young (18-30 yr) and old (80-89 yr) women.

The purpose of this study was to investigate mRNA expression of several key skeletal muscle myogenic controllers; myogenic differentiation factor (MyoD), muscle regulatory factor 4 (MRF4), myogenic factor 5 (Myf5), myogenin, myostatin, and myocyte enhancer factor 2 (MEF2) at rest and 4 h after a single bout of resistance exercise (RE) in young and old women. Eight young women (YW; 23 +/- 2 yr, 67 +/- 5 kg) and six old women (OW; 85 +/- 1 yr, 67 +/- 4 kg) performed 3 sets of 10 repetitions of bilateral knee extensions at 70% of one repetition maximum. Muscle biopsies were taken from the vastus lateralis before and 4 h after RE. Using real-time RT PCR, mRNA from the muscle samples was amplified and normalized to GAPDH. At rest, OW expressed higher (P < 0.05) levels of MyoD, MRF4, Myf5, myogenin, and myostatin compared with YW. In response to RE, there was a main time effect (P < 0.05) for the YW and OW combined in the upregulation of MyoD (2.0-fold) and MRF4 (1.4-fold) and in the downregulation of myostatin (2.2-fold). There was a trend (P = 0.08) for time x age interaction in MRF4. These data show that old women express higher myogenic mRNA levels at rest. The higher resting myogenic mRNA levels in old women may reflect an attempt to preserve muscle mass and function. When challenged with RE, old women appear to respond in a similar manner as young women.     

Time course of molecular responses of human skeletal muscle to acute bouts of resistance exercise.

Resistance exercise (RE) training, designed to induce hypertrophy, strives for optimal activation of anabolic and myogenic mechanisms to increase myofiber size. Clearly, activation of these mechanisms must precede skeletal muscle growth. Most mechanistic studies of RE have involved analysis of outcome variables after many training sessions. This study measured molecular level responses to RE on a scale of hours to establish a time course for the activation of myogenic mechanisms. Muscle biopsy samples were collected from nine subjects before and after acute bouts of RE. The response to a single bout was assessed at 12 and 24 h postexercise. Further samples were obtained 24 and 72 h after a second exercise bout. RE was induced by neuromuscular electrical stimulation to generate maximal isometric contractions in the muscle of interest. A single RE bout resulted in increased levels of mRNA for IGF binding protein-4 (84%), MyoD (83%), myogenin (approximately 3-fold), cyclin D1 (50%), and p21-Waf1 (16-fold), and a transient decrease in IGF-I mRNA (46%). A temporally conserved, significant correlation between myogenin and p21 mRNA was observed (r = 0.70, P < or = 0.02). The mRNAs for mechano-growth factor, IGF binding protein-5, and the IGF-I receptor were unchanged by RE. Total skeletal muscle RNA was increased 72 h after the second serial bout of RE. These results indicate that molecular adaptations of skeletal muscle to loading respond in a very short time. This approach should provide insights on the mechanisms that modulate adaptation to RE and may be useful in evaluating RE training protocol variables with high temporal resolution.     

Gender related and dexamethasone induced differences in the mRNA levels of the MRF genes in rat anterior tibial skeletal muscle

Muscle formation and postnatal growth is under the control of the muscle regulatory factors (MRF) gene family, consisting of four genes: MyoD1, myogenin, myf-5, and myf-6. Muscle mass is also known to be affected by specific drugs, like glucocorticoids. Glucocorticoids have also been characterized as muscle atrophying agents. However, glucocorticoids are also the only drugs reported to have a beneficial effect on the treatment of muscle degenerative disorders. Since muscle mass relates to gender, this may be partially caused by gender. The aim of this study is to investigate gender-related basal and dexamethasone-induced expression of the MRF genes. Gender-specific MRF mRNA levels were investigated in anterior tibial muscles of the rat. Myogenin, myf-5, and myf-6 mRNA level was significantly higher in female rats than in male rats. Since muscle mass is usually higher in males, we conclude that the development of gender-related differences in muscle mass is not primarily under the control of the mRNA levels of the MRF genes. Male rats treated with dexamethasone for 14 days (1 mg per kg body weight) showed increased levels of MyoD1, myogenin and myf-5 compared to control male rats. Female rats treated with dexamethasone showed decreased expression of myf-6 compared to control female rats. These results suggest that dexamethasone increase satellite cell-specific MRF activity in male muscle tissue, which is suggested to be associated with muscle hypertrophy, while maintenance of muscle tissue is affected in female muscle tissue. Therefore, we conclude that both basal and dexamethasone-induced MRF gene mRNA levels are regulated gender-specific.     

Induction of endogenous myosin light chain 1 and cardiac alpha-actin expression in L6E9 cells by MyoD1.

Rat L6E9 muscle cells commit to terminal differentiation by forming a large muscle syncitia complete with the expression of a large number of muscle-specific contractile protein genes. To determine whether these cells, which fail to synthesize MLC (myosin light chain) 1 and cardiac alpha-actin, exhibit a deficiency in the expression of muscle determination genes, we measured expression of MyoD1, myogenin, Myf-5, and MRF-4. Results show these cells do not synthesize MyoD1, yet express the other myogenic determination genes. Transient expression of exogenous MyoD1 in these cells is sufficient to activate endogenous MLC 1 and cardiac alpha-actin mRNA synthesis during muscle differentiation. Previously undetected myosin heavy chain (MHC) isoforms (beta-MHC and perinatal MHC) are also transcribed at low levels in L6E9 muscle cells, and in MyoD1-transfected L6E9 cells no change occurs in their expression. Furthermore, treatment with the demethylating agent 5-azacytidine activates expression of the endogenous MyoD1 gene in L6E9 cells and, subsequently, rescues deficiencies in their myogenic biochemical program. These results demonstrate that the endogenous MyoD1 gene in L6E9 cells is not defective and can be functionally activated. Also, the MyoD1 protein plays an essential role, which cannot be compensated by other known muscle determination proteins, in the induction of MLC 1 and cardiac alpha-actin expression.